CB2 receptors blocks accumulation of human hepatic myofibroblasts: a novel antifibrogenic pathway in the liver

ABSTRACT

Methods and compositions for treating diseases mediated by CB2 receptors are disclosed, including fibrosis associated with liver injury.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/508,178, filed Oct. 1, 2003, which is incorporated herein byreference in its entirety for all purposes.

BACKGROUND

Liver fibrosis is the common response to chronic liver injury,ultimately leading to cirrhosis and its complications. The fibrogenicprocess is consecutive to intense proliferation and accumulation ofhepatic myofibroblasts that synthesize fibrosis components andinhibitors of matrix degradation [1]. Hepatic myofibroblasts play a keyrole in the development of liver fibrosis associated with chronic liverdiseases, and their removal by apoptosis contributes to the resolutionof liver fibrosis. Better understanding of the molecular mechanisms andsignaling pathways that govern hepatic myofibroblast functions is aprerequisite for the identification of antifibrotic targets that willenable to develop liver-directed antifibrotic drugs. Along these lines,currently recognized antifibrotic strategies include: (i) reduction ofhepatic myofibroblast accumulation by molecules that block theirproliferation or stimulate their apoptosis, and/or (ii) reduction offibrosis by agents that inhibit extracellular matrix synthesis orenhance its degradation.

Cannabinoids are the main constituent of marijuana and includepsychoactive molecules, such as (−) Δ⁹-tetrahydrocannabinol (THC), andnon psychoactive substances, like cannabidiol. Endogenous naturalcannabinoids have also been characterized, anandamide and 2-arachidonylglycerol ([(5Z,8Z,11Z,14Z)-5,8,11,14-Eicosatetraenoic acid,2-hydroxy-1-(hydroxymethyl)ethyl ester]), which are arachidonicacid-derived lipids [2, 23]. Cannabinoids display analgesic, antiemetic,vasorelaxing and anti-inflammatory properties, and stimulate food intake[3; 2]. They also exert antitumoral effects, mainly due to theirantiproliferative and apoptotic properties [4]. Cannabinoid effects aremediated by activation of specific G protein-coupled receptors, CB1 andCB2 [5]. CB1 receptors are predominant in brain and are responsible forcannabinoid psychoactivity, whereas the peripheral CB2 receptors aremainly expressed in the immune system and are devoid of cannabinoidpsychoactive effects [5]. Atypical CB receptors, distinct from CB1 andCB2 have been described in brain and in vascular endothelial cells.

SUMMARY

There are only few data concerning the hepatic action of cannabinoids.CB1 and CB2 receptors are not expressed in hepatocytes [6]. However, CB1receptors are present in endothelial cells from hepatic artery, andtheir number increase in the cirrhotic liver [7]. The results presentedherein demonstrate for the first time the hepatic expression of CB2receptors in patients with chronic liver diseases, and the up-regulationof CB2 receptors in hepatic myofibroblasts. The results also show thatCB2 receptors trigger potent growth inhibitory and apoptotic effects,two major antifibrogenic properties of hepatic myofibroblasts. Growthinhibition is mediated by induction of cyclooxygenase-2, and apoptosisresults from CB2-dependent oxidative stress. These results indicatethat, during chronic liver injury, activation of CB2 receptors can limitfibrogenesis originating from chronic liver diseases of any etiology(alcoholic, viral, toxic) by blocking accumulation of hepaticmyofibroblasts.

Based upon these results, a variety of methods and compositions (e.g.,pharmaceutical compositions) have been developed for treating diseasesthat are mediated by the activity or expression of CB2 receptors,including fibrosis associated with liver injury and disease. The methodsare based, in part, on the finding that activation of CB2 receptors canreduce liver fibrogenesis associated with liver injury or disease. Thus,methods are provided for treating any hepatic diseases which result inhepatic fibrosis. The hepatic diseases include, but are not limited to,alcoholic liver cirrhosis, chronic viral hepatitis, non alcoholicsteatohepatitis and primary liver cancer.

Some methods that are provided are methods of treating fibrosis duringchronic liver injury and involves the use of cannabinoids. Othertreatment methods involve the use of agonists of CB2 receptor. Stillother methods involve the activation of CB2 receptors. Certain fibrosistreatment methods involve the up-regulation of CB2 receptors.

Also disclosed are methods for treating fibrosis during chronic liverinjury which involve administering an effective amount of a cannabinoidto a patient having a liver injury. Other treatment methods involveadministering an agent that activates a CB2 receptor to a patient havinga liver injury. Still other methods of treating liver diseases compriseadministering a composition comprising a non-selective agonist of CB2and a selective antagonist of CB1 to a patient having a liver disease.

Screening methods to identify novel ligands (e.g., agonists) for CB2receptors are also provided. Some of these screening methods involve (a)culturing hepatic myofibroblasts, (b) exposing the culture to acandidate ligand, (c) assessing the effect of the candidate ligands byan apoptosis, cell viability or cell proliferation assay, and (d)selecting a candidate ligand that has an effect in the assay.

Various compositions useful for treating fibrosis are also provided.Some compositions of this type contain one or more agonists of a CB2receptor. The composition can also include a pharmaceutically acceptablecarrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Expression of CB2 receptor protein during chronic liverdiseases. Representative distribution of CB2 receptor immunostaining onliver tissue sections obtained from biopsies of (A) normal liver and (B)active cirrhosis (Magnification×200). Arrows indicate representativeimmunostaining of mesenchymal cells within and at the edge of thefibrotic septa. Inset shows negative control staining on activecirrhosis obtained after preadsorption of the anti-CB2 antibody(directed against CB2 receptor blocking peptide) with the CB2 syntheticpeptide (Magnification×200). (C) Representative double immunostainingfor CB2 receptor (brown color) and smooth muscle alpha actin (red color)in cirrhotic liver (Magnification×630). Arrows indicate cellsimmuno-positive for both CB2 receptor and smooth muscle alpha actin.

FIG. 2: CB2 receptors are expressed and functional in cultured humanhepatic myofibroblasts. (A) Expression of CB2 receptor mRNA. RT-PCR forCB2 receptors was performed as described under “Methods”. The PCRproducts were then size-fractionated, blotted and the membranes werehybridized with a labeled oligonucleotide complementary to the CB2receptor sequences within the cDNA flanked by the PCR primers. Bandswith 223 bp corresponding to the size of the CB2 receptor product wasidentified. (B) Expression of CB2 receptor protein. CB2 receptor proteinwas detected by immunofluorescence as described under “Methods”(Magnification×630). (C) THC and JWH-015 enhance GTPγS binding in humanhepatic myofibroblasts and CHO-CB2 cells. Membranes from human hepaticmyofibroblasts or CHO-CB2 were assayed for [³⁵S]GTPγS binding assays asdescribed under ‘Methods’ with varying concentrations of THC. Resultsrepresent the mean±S.E.M of seven to nine experiments and are expressedas percent of control. Inset shows stimulation of [³⁵S]GTPγS binding by300 nM of THC or JWH-015 in human hepatic myofibroblasts. P<0.001 by2-way ANOVA for agonists treatments.

FIG. 3: Cannabinoids inhibit DNA synthesis in human hepaticmyofibroblasts via a CB2 receptor-dependent pathway. (A) Effects ofcannabinoids on DNA synthesis. Confluent human hepatic myofibroblastswere made quiescent by incubation in serum-free medium over 3 days.Cells were stimulated for 30 hrs with varying concentrations of THC,JWH-015, cannabidiol and ACEA, in the presence of 20 ng/ml of PDGF(platelet-derived growth factor). [³H]Thymidine incorporation into DNAwas measured as described under “Methods”. Results represent themean±SEM of 3 to 6 experiments and are expressed as percent of control.p<0.05 compared to control. Inset: Effects of selective CB1 CB2 receptoragonists on cAMP production in CHO cells overexpressing CB1 or CB2receptor. Confluent quiescent CHO cells overexpressing CB1 or CB2receptors were exposed for 10 min to 100 nM of JWH015, ACEA andcannabidiol or the respective concentrations of vehicle (ethanol forACEA and JWH015, methanol for cannabidiol). Cyclic AMP was extracted andmeasured as described under “Methods”. (B) Inhibition of DNA synthesisby THC is blocked by a CB2 receptor antagonist. Confluent quiescentcells were pretreated for 1 hr with 1 μM of the CB2 receptor antagonistSR144528 or vehicle. DNA synthesis was then measured as described in(A). Results represent the mean±SEM of 6 experiments. p<0.05 for THC vsvehicle and for THC+SR144528 vs THC.

FIG. 4: Cannabinoids trigger human hepatic myofibroblast death by anapoptotic mechanism. (A) Phase-contrast analysis (Magnification×100).Serum-deprived cells were incubated for 16 h with 4 μm THC or vehicle.(B) DAPI staining of the nuclei (Magnification×630) Serum-deprived cellswere incubated for 16 h with 4 μm THC or vehicle. (C) Caspase-3-likeactivity. Caspase-3-like activity kinetic was assayed on cell lysates atthe indicated time point as described under “Methods.” (D) DNA ladderformation. DNA was extracted and analyzed by electrophoresis on a 2%agarose gel stained with SYBR Green I. Serum-deprived cells werepreincubated for 1 h with 50 μM ZVAD or vehicle and further incubatedfor 16 h in the presence of 50 μM ZVAD or vehicle, together with either4 μM of THC or vehicle.

FIG. 5: Cannabinoids triggers apoptosis via a CB2 receptor-dependentpathway. (A) Effects of THC and CB1 and CB2 receptor selective agonistson hepatic myofibroblast viability. Serum-deprived cells were incubatedwith varying concentrations of THC, JWH015, cannabidiol and ACEA for 16hours. Cell viability was determined as described under “Experimentalprocedures”. Results are the mean +/− S.E.M. of 3 to 6 experiments(p<0.05 compared with vehicle). (B) Apoptosis triggered by THC isblocked by a CB2 receptor antagonist. Serum-deprived cells werepreincubated for 1 h with 2 μM of SR144528 or vehicle, and furtherincubated with varying concentrations of THC for 16 hours. Cellviability was determined as described under “Experimental procedures”.Results are the mean +/− S.E.M. of 6 experiments (p<0.05 for THC vsvehicle and for THC+SR144528 vs THC). Inset: Effects of SR144528 oncaspase-3 activation by THC. Serum-deprived cells were preincubated for1 h with 2 μM of SR144528 or vehicle, and further incubated with 4 μM ofTHC or vehicle for 5 hours. Caspase-3-like activity was assayed onlysates, as described under “Methods”. Results are the mean +/− S.E.M of5 experiments (p<0.05 for THC vs vehicle and for THC+SR144528 vs THC).SR144528 alone had no effect either on cell viability or caspase-3 likeactivity.

FIG. 6: Growth inhibitory and apoptotic effects of THC are mediated bytwo distinct signaling pathways. (A) COX-2 mediates inhibition of humanhepatic myofibroblast proliferation by THC. Cells were pretreated for 1h with 10 μM NS-398 or vehicle, further stimulated with 20 ng/ml PDGF-BBand varying concentrations of THC, and DNA synthesis was measured as inFIG. 3. Results (mean±SEM, n=3-5) are expressed as percent of respectivecontrol (17000±6000 cpm for PDGF-BB; 34000±12000 cpm forPDGF-BB+NS-398). p<0.05 for NS-398 effects. Inset: Cells were pretreatedfor 1 h with 5 mM NAC, 25 μM EUK8 or vehicle and further stimulated 20ng/ml PDGF-BB and 750 nM THC. (B) THC induces COX-2 and stimulates COXactivity in human hepatic myofibroblasts. Western blot analysis of COX-2protein induction in extracts of cells treated with varyingconcentrations of THC for 8 h (n=3). A typical blot is shown. Resultswere normalized relative to B-actin expression. COX activity was assayedafter 8 h incubation with 1000 nM THC. At the end of incubation, 10 μMof arachidonic acid was added for 30 min and PGE2 released in thesupernatants was measured as described under “Methods”. Results are themean of sextuplate determinations obtained from 2 experiments (# p<0.05vs control).s. (C) THC-induced apoptosis involves ROS production.Serum-deprived hepatic myofibroblasts were pretreated for 1 hr witheither 5 mM NAC (N-Acetyl Cysteine), 10 μM EUK8 or vehicle. Caspase3-like activity was assayed as in FIG. 4, after a 5 hr treatment with 4μM THC or vehicle (mean±SEM, n=3; # p<0.05 vs THC). Antioxidants addedalone had no effect on caspase-3-like activity. Maximal increase ofcaspase-3 activity by THC was 6.3 ±2 fold (100%). (D) Effect of THC onthe production of reactive oxygen species (ROS). Human hepaticmyofibroblasts were loaded with DCFH-DA (dichlorofluoroscein diacetate)for 20 min at 37° C., together with 3 μM THC and the fluorescence wasmonitored in a FL-600 fluorimeter. Results are the mean +/− SEM of 6experiments. # P<0.05 for THC vs basal.

DETAILED DESCRIPTION

I. Definitions

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULARBIOLOGY (2d ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE ANDTECHNOLOGY (Walker ed., 1988); THE GLOSSARY OF GENETICS, 5TH ED., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, THEHARPER COLLINS DICTIONARY OF BIOLOGY (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

Various biochemical and molecular biology methods are well known in theart. For example, methods of isolation and purification of nucleic acidsare described in detail in WO 97/10365, WO 97/27317, Chapter 3 ofLaboratory Techniques in Biochemistry and Molecular Biology:Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic AcidPreparation, (P. Tijssen, ed.) Elsevier, N.Y. (1993); Chapter 3 ofLaboratory Techniques in Biochemistry and Molecular Biology:Hybridization With Nucleic Acid Probes, Part 1. Theory and Nucleic AcidPreparation, (P. Tijssen, ed.) Elsevier, N.Y. (1993); and Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,N.Y., (1989); Current Protocols in Molecular Biology, (Ausubel, F. M. etal., eds.) John Wiley & Sons, Inc., New York (1987-1993). Large numbersof tissue samples can be readily processed using techniques known in theart, including, for example, the single-step RNA isolation process ofChomczynski, P. described in U.S. Pat. No. 4,843,155.

As used herein, references to specific proteins (e.g., CB1 and CB2) caninclude a polypeptide having a native amino acid sequence, as well asvariants and modified forms regardless of origin or mode of preparation.A protein that has a native amino acid sequence is a protein having thesame amino acid sequence as obtained from nature (e.g., a naturallyoccurring CB1 or CB2). Such native sequence proteins can be isolatedfrom nature or can be prepared using standard recombinant and/orsynthetic methods. Native sequence proteins specifically encompassnaturally occurring truncated or soluble forms, naturally occurringvariant forms (e.g., alternatively spliced forms), naturally occurringallelic variants and forms including postranslational modifications. Anative sequence protein includes proteins following post-translationalmodifications such as glycosylation of certain amino acid residues.

Variants refer to proteins that are functional equivalents to a nativesequence protein that have similar amino acid sequences and retain, tosome extent, one or more activities of the native protein. Variants alsoinclude fragments that retain activity. Variants also include proteinsthat are substantially identical (e.g., that have 80, 85, 90, 95, 97,98, 99%, sequence identity) to a native sequence. Such variants includeproteins having amino acid alterations such as deletions, insertionsand/or substitutions. A “deletion” refers to the absence of one or moreamino acid residues in the related protein. The term “insertion” refersto the addition of one or more amino acids in the related protein. A“substitution” refers to the replacement of one or more amino acidresidues by another amino acid residue in the polypeptide. Typically,such alterations are conservative in nature such that the activity ofthe variant protein is substantially similar to a native sequenceprotein (see, e.g., Creighton (1984) Proteins, W.H. Freeman andCompany). In the case of substitutions, the amino acid replacing anotheramino acid usually has similar structural and/or chemical properties.Insertions and deletions are typically in the range of 1 to 5 aminoacids, although depending upon the location of the insertion, more aminoacids can be inserted or removed. The variations can be made usingmethods known in the art such as site-directed mutagenesis (Carter, etal. (1986) Nucl. Acids Res. 13:4331; Zoller et al. (1987) Nucl. AcidsRes. 10:6487), cassette mutagenesis (Wells et al. (1985) Gene 34:315),restriction selection mutagenesis (Wells, et al. (1986) Philos. Trans.R. Soc. London SerA 317:415), and PCR mutagenesis (Sambrook et al.,Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring HarborPress, N.Y., (2001)).

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., Current Protocols In Molecular Biology, GreenePublishing and Wiley-Interscience, New York (supplemented through 1999).Each of these references and algorithms is incorporated by referenceherein in its entirety. When using any of the aforementioned algorithms,the default parameters for “Window” length. gap penalty, etc., are used.One example of algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).

Modified forms of a protein generally refer to proteins in which one ormore amino acids of a native sequence have been altered to anon-naturally occurring amino acid residue. Such modifications can occurduring or after translation and include, but are not limited to,phosphorylation, glycosylation, cross-linking, acylation and proteolyticcleavage.

The term “CB2 receptor” has its general meaning in the art [5], and mayinclude naturally occurring CB2 receptors and variants and modifiedforms thereof. The term may also refer to fusion proteins in which adomain from CB2 that retains at least one CB2 activity is fused, forexample, to another polypeptide (e.g., a polypeptide tag such as areconventional in the art). The CB2 receptor can be from any source, buttypically is a mammalian (e.g., human and non-human primate) CB2,particularly a human CB2. An exemplary native CB2 amino acid sequence isprovided in Accession No NP_(—)001832.

CB2 activity” as used herein refers broadly to any biological activityassociated with CB2. Thus, the term includes the specific binding of aligand to CB2. The term also refers to various signal transducingactivities of the receptor. Representative activities of CB2 include,ability to bind agonists such as those listed herein, activation ofapoptosis, and inhibition of hepatic myofibroblast proliferation. Thus,the term “activate CB2” and other related terms refers to a processwhereby one or more activities of CB2 are promoted or induced.

The term “CB1 receptor” has its general meaning in the art [5], and mayinclude naturally occurring CB1 receptor and variants and modified formsthereof. The term also refers to fusion proteins in which a domain fromCB1 that retains at least one CB1 activity is fused, for example, toanother polypeptide (e.g., a polypeptide tag such as are conventional inthe art). The CB1 receptor can be from any source, but typically is amammalian (e.g., human and non-human primate) CB1, particularly a humanCB1. CB1 receptors include for example, two isoforms: a long isoform(Accession No NP_(—)057167) and a shorter one truncated in the NH2terminal part corresponding to a splice variant (Accession NoNP_(—)149421).

“CB1 activity” as used herein refers broadly to any biological activityassociated with CB1. Thus, the term includes the specific binding of aligand to CB1. The term also refers to various signal transducingactivities of the receptor. Representative activities of CB1 include,but are not limited to, ability to bind antagonists such as those listedherein and cannabinoid psychoactivity. Thus, the term “activate CB1” andother related terms refers to a process whereby one or more activitiesof CB1 are promoted or induced.

“Agonist” as used herein has its general meaning in the art, and refersto a compound natural or not which has the capability to activate areceptor. The term “selective CB2 receptor agonist” as used hereinrefers to a compound able to activate selectively CB2 receptors and notany other receptor such as CB1 receptors. The term “non selective CB2receptor agonist” as used herein refers to compound natural or not whichhas the capability to activate CB2 receptors but also CB1 receptors.

“Antagonist” as used herein has its general meaning in the art, andrefers to a compound natural or not which has the capability to inhibitthe activation of a receptor. A “selective CB1 antagonist” is hereindefined as a compound able to selectively inhibit the activation of CB1receptors and not any other receptor such as CB2 receptors.

The term “expression” when used in the context of expression of a geneor nucleic acid refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of a mRNA. Gene products also include RNAs whichare modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

The terms “up-regulated and “activation” when used in reference to theexpression of a nucleic acid such as a gene (e.g., CB2 receptor) refersto any process which results in an increase in production of a geneproduct or activity of the gene product. A gene product can be eitherRNA (including, but not limited to, mRNA, rRNA, tRNA, and structuralRNA) or protein. Accordingly, gene up-regulation or activation includesthose processes that increase transcription of a gene and/or translationof a mRNA. Examples of gene up-regulation or activation processes thatincrease transcription include, but are not limited to, those thatfacilitate formation of a transcription initiation complex, those thatincrease transcription initiation rate, those that increasetranscription elongation rate, those that increase processivity oftranscription and those that relieve transcriptional repression (by, forexample, blocking the binding of a transcriptional repressor). Geneup-regulation or activation can constitute, for example, inhibition ofrepression as well as stimulation of expression above an existing level.Examples of gene up-regulation or activation processes that increasetranslation include those that increase translational initiation, thosethat increase translational elongation and those that increase mRNAstability.

The level of gene expression, including the level of gene activation orup-regulation, can be quantitated utilizing a number of establishedtechniques including, but not limited to, Northern-Blots, RNaseprotection assays (RPA), nucleic acid probe arrays, quantitative PCR(e.g., the so-called TaqMan assays), dot blot assays and in-situhybridization.

In general, gene up-regulation or activation comprises any detectableincrease in the production of a gene product, preferably an increase inproduction of a gene product by at least 30, 40, 50 or 100%, in otherinstances from about 2- to about 5-fold or any integer therebetween, instill other instances between about 5- and about 10-fold or any integertherebetween, sometimes between about 10- and about 20-fold or anyinteger therebetween, in other instances between about 20- and about50-fold or any integer therebetween, in yet other instances betweenabout 50- and about 100-fold or any integer therebetween, and in stillother instances 100-fold or more. The phrases up-regulation andactivation are typically assessed relative to a control or baselinelevel.

As used herein a “control” or “baseline value” generally refers to avalue (or ranges of values) against which an experimental or determinedvalue (e.g., one determined for a patient sample as part of a diagnosticor prognostic test) is compared. Thus, in the case of CB2 up-regulation,the baseline value can be a value for CB2 activity or expression for asample obtained from the same individual at a different time point. Inother instances, the baseline value is a value determined for a controlcell or individual, or a statistical value (e.g., an average or mean)established for a population of control cells or individuals. In thespecific instance of CB2 up-regulation, the control can be a cell,individual or populations thereof for which CB2 levels would not beexpected to be up-regulated. Thus, for instance, a control individual orcontrol population can include healthy individuals, particularly thosethat have no liver injury. The population that serves as a control canvary in size, having as few as a single member, but potentiallyincluding tens, hundreds, or thousands of individuals. When the controlis a large population, the baseline value can be a statistical valuedetermined from individual values for each member or a value determinedfrom the control population as an aggregate.

In the case of a screening assay, the control value can be a value for acontrol reaction that is conducted under conditions that are identicalthose of a test assay, except that the control reaction is conducted inthe absence of a candidate agent whereas the test assay is conducted inthe presence of the candidate agent. The control value can also be astatistical value (e.g., an average or mean) determined for a pluralityof control assays. The control assay(s) upon which the control value isdetermined can be conducted contemporaneously with the test orexperimental assay or can be performed prior to the test assay. Thus,the control value can be based upon contemporaneous or historicalcontrols.

A difference is typically considered to be “statistically significant”if in general terms an observed value differs from a control orbackground value by more than the level of experimental error. Adifference can be considered “statistically significant” if theprobability of the observed difference occurring by chance (the p-value)is less than some predetermined level. As used herein a “statisticallysignificant difference” refers to a p-value that is <0.05, preferably<0.01 and most preferably <0.001.

II. Overview

A variety of methods for treating fibrosis associated with liver injuryare provided. The methods are based, in part, upon the recognition thathepatic myofibroblasts are central for the development of liver fibrosisassociated with chronic liver diseases and that blocking theiraccumulation can prevent fibrogenesis. Cannabinoids act via tworeceptors, CB I, responsible for their psychoactive effects, and CB2,expressed in peripheral tissues. In liver biopsies from patients withactive cirrhosis of various etiologies, immunohistochemistry showed thepresence of CB2 receptors in non-parenchymal cells located within and atthe edge of fibrous septa. In contrast, no expression of CB2 receptorswas detected in normal human liver. CB2 receptors were expressed inhuman hepatic myofibroblasts, as shown by immunocytochemistry in liverbiopsies and in cultured cells, and functional, as evidenced in GTP-YSbinding assays. Activation of CB2 receptors led to growth inhibition andapoptosis of cultured human hepatic myofibroblasts via a CB2-dependentprocess, as demonstrated using selective CB2 agonists (JWH-015 andcannabidiol) and antagonist (SR 144528). The antiproliferative effect ofTHC was blunted by ibuprofen, a cyclooxygenase (COX) inhibitor and NS398 ([N-(2-cyclohexyloxy-4-nitrophenyl)methane sulfonamide] from BiomolResearch Labs (Plymouth Meeting, Pa.); see, e.g., Liu X. H., et al.(1998) Cancer Res. 58: 4245-4249), a selective COX-2 inhibitor.Accordingly, THC induced COX-2. In contrast, THC-induced apoptosis wasblocked by the antioxidants N-acetyl cysteine and EUK 8 (see, e.g,Pucheu, S., et al. (1996) Cardiovasc. Drugs Ther. 10:331-339), and THCgenerated production of reactive oxygen species.

The results demonstrate that the liver is a target of cannabinoidsduring chronic liver diseases. Indeed, CB2 receptors are up-regulated inhepatic myofibroblasts, and their activation reduces accumulation ofthese cells by triggering potent growth inhibitory and apoptoticeffects. These results provided herein thus indicate that activation ofCB2 receptors can limit liver fibrogenesis during chronic liver injury.The results also indicate that a CB2 receptor-based antifibroticstrategy can be used which is devoid of undesirable CB1-mediatedpsychotropic effects.

Compositions useful in treating various diseases associated with liverinjury are also provided, as are methods of identifying agents thatmodulate (e.g., increase) the activity of CB2 and which can be utilizedin the development of new pharmaceutical compositions.

III. Treatment Methods, Pharmaceutical Compositions and Methods ofAdministration

A. General

A variety of pharmaceutical compositions are provided. Some compositionscomprise one or more active ingredients that activate the CB2 receptoror up-regulate the expression of CB2 receptors. Certain compositionsthus include one or more agonists of the CB2 receptor. Thepharmaceutical compositions that are provided are useful in treatingvarious diseases that are mediated by CB2 receptors, including treatmentof fibrosis associated with liver injury (e.g., cirrhosis). The terms“treating” and “treatment” as used herein refer to reduction in severityand/or frequency of symptoms, elimination of symptoms and/or underlyingcause, prevention of the occurrence of symptoms and/or their underlyingcause, and improvement or remediation of damage.

B. Composition

The pharmaceutical compositions used for prophylactic or therapeutictreatment comprise an active therapeutic agent, for example, an agentthat activates the CB2 receptor or up-regulates the expression of CB2receptors. Examples of such agents include various agonists. Theagonists can be selective for CB2 or non-selective for CB2. One generalclass of agonists are the cannabinoids. Some compositions include aplurality of agonists (selective and/or non-selective). Still othercompositions that are provided include a combination of one or more CB2agonists and one or more CB1 antagonists.

One specific example of a selective agonist that can be utilized incertain compositions is palmitoylethanolamide[N-(2-Hydroxyethyl)hexadecanamide] (see, e.g., Facci, et al. (1995)Proc. Natl. Acad. Sci. USA 92:3376), which is commercially-availablefrom Tocris. The chemical structure is as follows:

The molecule referred to as JWH-133[(6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran]is another exemplary selective agonist that can be incorporated into acomposition (see, e.g., Huffman et. al. (1999) Bioorg. Med. Chem.7:2905). The structure of JWH-133 is provided below:

Other selective CB2 receptor agonists that can be used include thosedisclosed in published U.S. Patent Application U.S. 2004034090, entitled“3-Arylindole derivatives and their use as CB2 receptor agonists.”Agonists of this class have the following general structure:

-   -   in which:    -   Ar represents:        -   a) a phenyl mono-, di- or trisubstituted by one or more            groups chosen from: a halogen atom, a (C1-C4)alkyl, a            trifluoromethyl, an amino, a nitro, a hydroxyl, a            (C1-C4)alkoxy, a (C₁-C₄)alkylsulphanyl or a            (C1-C4)alkylsulphonyl;        -   b) a naphthyl which is unsubstituted or substituted once or            twice by a halogen atom, a (C1-C4)alkyl or a            trifluoromethyl;    -   A represents a C2-C6 alkylene radical;    -   Y represents a group chosen from SR4, SOR4, SO2R4, SO2NR5R6,        N(R7)SO2R4, OR4 or NR7SO2NR5R6;    -   R1, R3 and R′3 represent, each independently of one another,        hydrogen, a hydroxyl, a halogen atom, a (C1-C4)alkyl, a        trifluoromethyl or a (C1-C4)alkoxy;    -   R2 represents hydrogen or a (C1-C4)alkyl;    -   R4 represents a (C1-C4)alkyl or a trifluoromethyl    -   R5 and R6 each independently represent hydrogen or a        (C1-C4)alkyl; and    -   R7 represents hydrogen or a (C1-C4)alkyl.

Still other classes of agonists that can be included in the compositionsinclude, but are not limited to, those described in 1) U.S. Pat. Nos.6,013,648 and 5,605,906; 2) Published PCT applications WO0132169,WO0128497 and WO9618391; 3) published U.S. Patent Applications U.S.2004077643 and U.S. 2002173528; and 4) EP1374903, each of which isincorporated herein by reference in its entirety for all purposes.

Various non-selective agonists of CB2 can also be utilized in certainmethods and incorporated into pharmaceutical compositions. For instance,the composition can include CP 55,940[(−)-cis-3-[2-Hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol](Wiley et al (1995) Neuropharmacology 34:669), which is available fromTocris. The compound has the following structure:

Hu 210 is another one example of a suitable non-selective agonist. HU210 [(6aR)-trans-3-(11,1-Dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo[b,d]pyran-9-methanol](Mechoulam et al (1988) 44 762) has the following structure:

Another useful non-selective agonist of CB2 is WIN 55,212-2[(R)-(+)-[2,3-Dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanonemesylate] (Martellotta et al (1998) Neuroscience 85 327). The structureof WIN 55,212-2 is as follows:

Compositions that are provided herein can also incorporate Anandamide[N-(2-Hydroxyethyl)-5Z,8Z, 11Z,14Z-eicosatetraenamide] (Pertwee (1999)Curr. Med. Chem. 6 635), which has the structure as follows:

As noted above, some compositions include a non selective agonist of CB2receptor in combination with a selective antagonist of CB1 receptor.Such suitable antagonists of CB1 receptor include, but are not limitedto, SR141716, AM 281, AM 251, the substituted amides described inWO03/077847, the substituted aryl amides described in WO03/087037, thesubstituted imidazoles described in WO03/063781, bicyclic amidesdescribed in WO03/086288, the terphenyl derivatives described in WO03/084943, the aryl-benzo[b]thiophene and benzo[b]furan compoundsrespectively described in U.S. Pat. Nos. 5,596,106 and 5,747,524, theazetidine derivatives described in FR2805817, 3-amino-azetidinedescribed in FR2805810, or the 3-Substituted or 3,3-disubstituted1-(di-((hetero)aryl)-methyl)-azetidine derivatives described inFR2805818.

N-pipéridino-5-(4-chlorophényl)-1-(2,4-dichlorophényl)-4-methylpyrazole-3-carboxamide,known commercially as SR141716 or rimonabant, and its preparation aredescribed in the European patent application EP656354-A1 and isrepresented by the formula as follows:

N-(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-1H-pyrazole-3-carboxamide,known commercially as AM251, has the structure described below:

1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide,known commercially as AM281, has the structure as follows:

Certain compositions that are provided include at least one agonist ofCB2 receptor (selective or not) and at least one selective antagonist ofCB1 receptor. Some compositions, for example, comprise a least oneagonist of CB2 receptor (selective or not) in combination with SR141716.

The compositions, in addition to the active ingredient(s) such as thosejust listed, may also include, depending on the formulation desired,pharmaceutically-acceptable, non-toxic carriers or diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, buffered water, physiologicalsaline, PBS, Ringer's solution, dextrose solution, and Hank's solution.In addition, the pharmaceutical composition or formulation may alsoinclude other carriers, adjuvants, or non-toxic, nontherapeutic,nonimmunogenic stabilizers, excipients and the like. The compositionsmay also include additional substances to approximate physiologicalconditions, such as pH adjusting and buffering agents, toxicityadjusting agents, wetting agents, detergents and the like.

Further guidance regarding formulations that are suitable for varioustypes of administration can be found in Remington's PharmaceuticalSciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985).For a brief review of methods for drug delivery, see, Langer, Science249:1527-1533 (1990).

C. Administration

The compositions containing the CB2 agonist(s) can be administered forprophylactic and/or therapeutic treatments. The active ingredient (e.g.,CB2 receptor agonist(s)) in the pharmaceutical composition generally ispresent in an “effective amount.” By an “effective amount” of apharmaceutical composition is meant a sufficient, but nontoxic amount ofthe agent to provide the desired effect. The term refers to an amountsufficient to treat a subject (e.g., a mammal, particularly a human).Thus, the term “therapeutic amount” refers to an amount sufficient toremedy a disease state or symptoms, by preventing, hindering, retardingor reversing the progression of the disease or any other undesirablesymptoms whatsoever. The term “prophylactically effective” amount refersto an amount given to a subject that does not yet have the disease, andthus is an amount effective to prevent, hinder or retard the onset of adisease.

In therapeutic applications, compositions are administered to a patientalready suffering from a disease, as just described, in an amountsufficient to cure or at least partially arrest the symptoms of thedisease and its complications. An appropriate dosage of thepharmaceutical composition is readily determined according to any one ofseveral well-established protocols. For example, animal studies (e.g.,mice, rats) are commonly used to determine the maximal tolerable dose ofthe bioactive agent per kilogram of weight. In general, at least one ofthe animal species tested is mammalian. The results from the animalstudies can be extrapolated to determine doses for use in other species,such as humans for example. What constitutes an effective dose alsodepends on the nature and severity of the disease or condition, and onthe general state of the patient's health.

In prophylactic applications, compositions containing, for example CB2receptor agonists, are administered to a patient susceptible to orotherwise at risk of a disease mediated by CB2 receptors (e.g. fibrosisformation). Such an amount is defined to be a “prophylacticallyeffective” amount or dose. In this use, the precise amounts againdepends on the patient's state of health and weight.

In both therapeutic and prophylactic treatments, the agonist containedin the pharmaceutical composition can be administered in several dosagesor as a single dose until a desired response has been achieved. Thetreatment is typically monitored and repeated dosages can beadministered as necessary. Compounds of the invention may beadministered according to dosage regimens established wheneveractivation of CB2 receptors is required.

The daily dosage of the products may be varied over a wide range from0.01 to 1,000 mg per adult per day. Preferably, the compositions contain0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250and 500 mg of the active ingredient for the symptomatic adjustment ofthe dosage to the patient to be treated. A medicament typically containsfrom about 0.01 mg to about 500 mg of the active ingredient, preferablyfrom 1 mg to about 100 mg of the active ingredient. An effective amountof the drug is ordinarily supplied at a dosage level from 0.0002 mg/kgto about 20 mg/kg of body weight per day, especially from about 0.001mg/kg to 7 mg/kg of body weight per day. It will be understood, however,that the specific dose level and frequency of dosage for any particularpatient may be varied and will depend upon a variety of factorsincluding the activity of the specific compound employed, the metabolicstability, and length of action of that compound, the age, the bodyweight, general health, sex, diet, mode and time of administration, rateof excretion, drug combination, the severity of the particularcondition, and the host undergoing therapy.

The pharmaceutical compositions described herein can be administered ina variety of different ways. Examples include administering acomposition containing a pharmaceutically acceptable carrier via oral,intranasal, rectal, topical, intraperitoneal, intravenous,intramuscular, subcutaneous, subdermal, transdermal, and intrathecalmethods.

For oral administration, the active ingredient can be administered insolid dosage forms, such as capsules, tablets, and powders, or in liquiddosage forms, such as elixirs, syrups, and suspensions. The activecomponent(s) can be encapsulated in gelatin capsules together withinactive ingredients and powdered carriers, such as glucose, lactose,sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesiumstearate, stearic acid, sodium saccharin, talcum, magnesium carbonate.Examples of additional inactive ingredients that may be added to providedesirable color, taste, stability, buffering capacity, dispersion orother known desirable features are red iron oxide, silica gel, sodiumlauryl sulfate, titanium dioxide, and edible white ink. Similar diluentscan be used to make compressed tablets. Both tablets and capsules can bemanufactured as sustained release products to provide for continuousrelease of medication over a period of hours. Compressed tablets can besugar coated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric-coated for selectivedisintegration in the gastrointestinal tract. Liquid dosage forms fororal administration can contain coloring and flavoring to increasepatient acceptance.

If desired, it is possible to formulate solid or liquid formulations inan enteric-coated or otherwise protected form. In the case of liquidformulations, the formulation can be mixed or simply coadministered witha protectant, such as a liquid mixture of medium chain triglycerides, orthe formulation can be filled into enteric capsules (e.g., of soft orhard gelatin, which are themselves optionally additionally entericcoated). Alternatively, solid formulations comprising the polypeptidecan be coated with enteric materials to form tablets. The thickness ofenteric coating on tablets or capsules can vary. Typical thickness rangefrom 0.5 to 4 microns in thickness. The enteric coating may comprise anyof the enteric materials conventionally utilized in orally administrablepharmaceutical formulations. Suitable enteric coating materials areknown, for example, from Remington's Pharmaceutical Sciences, MacePublishing Company, Philadelphia, 17th ed. (1985); and Hagars Handbuchder Pharmazeutischen Praxie, Springer Verlag, 4^(th) ed., Vol. 7a(1971).

Another delivery option involves loading the composition intolipid-associated structures (e.g., liposomes, or other lipidiccomplexes) which may enhance the pharmaceutical characteristics of thepolypeptide component of the composition. The complex containing thecomposition may subsequently be targeted to specific target cells by theincorporation of appropriate targeting molecules (e.g., specificantibodies or receptors). It is also possible to directly complex thepolypeptide with a targeting agent.

Compositions prepared for intravenous administration typically contain100 to 500 ml of sterile 0.9% NaCl or 5% glucose optionally supplementedwith a 20% albumin solution and 100 to 500 mg of a polypeptide of theinvention. Methods for preparing parenterally administrable compositionsare well-known in the art and described in more detail in varioussources, including, for example, Remington's Pharmaceutical Science,Mack Publishing, Philadelphia, Pa., 17th ed., (1985).

Particularly when the compositions are to be used in vivo, thecomponents used to formulate the pharmaceutical compositions of thepresent invention are preferably of high purity and are substantiallyfree of potentially harmful contaminants (e.g., at least National Food(NF) grade, generally at least analytical grade, and more typically atleast pharmaceutical grade). Moreover, compositions intended for in vivouse are usually sterile. To the extent that a given compound must besynthesized prior to use, the resulting product is typicallysubstantially free of any potentially toxic agents, particularly anyendotoxins, which may be present during the synthesis or purificationprocess. Compositions for parental administration are also sterile,substantially isotonic and made under GMP conditions.

IV. Methods for Identifying CB2 Modulators

A variety of screening methods can be utilized to identify modulators(e.g., agonists or antagonists) of CB2. Because the current inventorshave found that activation of CB2 activity plays an important role ininhibiting the growth of human hepatic myofibroblasts, agonistsidentified in the screening methods can be used, for example, ascandidate agents in the treatment of undesirable fibrosis associatedwith liver disease.

A. Binding Assays

Competition binding assays can also be used in the screening methods. Inassays of this type, a known ligand of CB2 such as those listed above,is combined with CCR1 (or a variant or fragment thereof that retainsbinding activity) in the presence of a test agent. The extent of bindingbetween the known ligand and CB2 in the presence of the test agent iscompared with the level of ligand binding in a control, typically asimilar assay conducted in the absence of the test agent. A difference(e.g., a statistically significant difference) between the test andcontrol assays is an indication that the test agent is a modulator ofCB2 activity. An increase in binding of the known ligand is anindication that the test agent is an agonist. A decrease in binding ofthe known ligand, in contrast, is an indication that the test agent isan antagonist.

The binding assays can be conducted as cell-based assays, which usecells that naturally express CB2 (e.g., cultured hepatic cells) or cellsthat have been stably or transiently transfected and thus express CB2.The cells are maintained under conditions appropriate for expression ofthe receptor and are contacted with the test agent and the known ligandunder conditions appropriate for binding to occur. Binding can bedetected using standard techniques. For example, the extent of bindingcan be determined relative to a suitable control (for example, relativeto background in the absence of a putative agent, or relative to a knownligand). Optionally, a cellular fraction, such as a membrane fraction,containing the receptor can be used in lieu of whole cells. Detection ofbinding or complex formation can be detected directly or indirectly. Forexample, the test agent or the known ligand can be labeled with asuitable label (e.g., fluorescent label, chemiluminescent label, isotopelabel, enzyme label, and the like) and binding can be determined bydetection of the label.

Other binding assays, however, are non-cellular assays. Such assays canbe conducted by immobilizing CB2 to a support, for example, and thecontacting the immobilized receptor with a composition containing thetest agent. Formation of complex can be detected and optionallyquantified as just described. The CB2 protein in such assays may be afusion protein that includes a CB2 domain that retains an activity ofCB2 and a tag (e.g., any of the polypeptide tags listed supra). In theseassays, the fusion protein is immobilized to a support via the tag(e.g., an antibody deposited on the support that binds the tag). Furtherguidance regarding receptor binding assays is provided, for example, byParce et al., 1989, Science 246: 243-247; and Owicki et al., 1990, Proc.Nat'l Acad. Sci. USA 87: 4007-4011.

B. Biological Assays

Other screening assays that are provided are designed not only todetermine whether a test agent binds CB2, but also determine if the testagent can modulate a CB2 activity. Because CB2 is a G-protein coupledreceptor, the binding of a ligand to CB2 can result in signaling, andthe activity of G proteins as well as other intracellular signalingmolecules can be stimulated. Examples of biological activities mediatedby CB2 include stimulation of apoptosis and cell death, and inhibitionof cell growth and cell proliferation. The induction of a biologicalfunction by a test agent can be monitored using any suitable method. Thecapacity of a test agent to modulate the activity of CCR1 can bedetermined in the presence of a ligand. The examples provide furtherdetails on certain (e.g., apoptosis) assays that can be utilized in thescreening methods.

1. Exemplary Cell Proliferation Assays

Cellular proliferation assays can be conducted in a variety of differentways, including, for example: actual cell counting, clonogenic assays,measuring metabolic activity, measuring DNA synthesis and/or measuringthe level of molecules that regulate cell cycle (e.g., CDK kinaseassays). A brief summary of these approaches follows. For a generalreview of some of these approaches, see for example, Roche MolecularBiochemicals, “Apoptosis and Cell Proliferation”, 2^(nd) Revisededition, pages 66-114, which is incorporated herein by reference in itsentirety for all purposes. Regardless of the particular approach takenfor determining cell proliferation, certain screening methods thatinvolve monitoring cell proliferation involve contacting a cell or cellpopulation expressing CB2 in the presence of a test agent (optionally inthe presence of a known CB2 ligand) and then determining the level ofcell proliferation in the presence of the test compound. The determinedlevel of cell proliferation is then compared with the level of cellproliferation in the absence of the test agent. An increase in cellproliferation in the presence of the test agent indicates that the testagent is an inhibitor of CB2, whereas a decrease in cell proliferationindicates that the test agent is an activator of CB2.

One approach to detect cell proliferation is simply to count the numberof cells using a cell counting device such as a hemacytometer. In theclonogenic assay approach, a defined number of cells are plated out ontoa suitable media and the number of colonies that are formed after adefined period of time are determined. The clonogenic approach can besomewhat cumbersome for large number of samples and for cells thatdivide only a few times and then become quiescent.

A number of different assays for measuring metabolic activity areavailable. One approach is to incubate the cells with a tetrazolium salt(e.g., MTT, XTT or WST-1), which becomes cleaved during cellularmetabolism to form a colored formazan product. Further guidanceregarding assays of this type are provided by Cook, J. A. and Mitchell,J. B. (1989) Anal. Biochem. 179:1; Roehm, N. W. et al. (1991) J.Immunol. Methods 142:257; Slater, T. F., et al. (1963) Biochem. Biophys.Acta 77:383; Berridge, M. V. and Tan, A. S. (1993) Arch. Biochem.Biophys. 303:474; Cory, A. H., et al. (1991) Cancer Commun. 3:207;Jabbar, S. A. B., et al. (1989) Br. J. Cancer 60: 523; and Scudiero, E.A., et al. (1988) Cancer Res. 48, 4827, each of which is incorporatedherein by reference in its entirety for all purposes. A variety of kitsfor performing such assays are available from Roche MolecularBiochemicals. Other assays in this class involve the measurement of ATPand involve detecting the formation of luminescence formed via theactivity of luciferase. Such assays are commercially available fromPerkin Elmer (see, e.g., ATPlite™ Assay kits).

Because DNA is replicated during cell proliferation, assays that providea measure of DNA replication also provide an useful measure of cellproliferation. Assays of this type typically involve adding labeled DNAprecursors to a cell culture. Cells that are about to divide incorporatethe labeled nucleotide into their DNA. Some approaches utilize tritiatedthymidine ([3H]-TdR) and measure the amount of incorporated tritiatedthymidine using liquid scintillation counting. To avoid usingradioactive compounds, other assays utilize the thymidine analog5-bromo-2′deoxy-uridine (BrdU), which becomes incorporated into DNA justlike thymidine. Incorporated BrdU can be detected quantitatively using acellular immunoassay that utilizes monoclonal antibodies directedagainst BrdU. Commercial kits for performing such assays are availablefrom a number of sources including Roche Molecular Biochemicals.

2. Apoptosis/Cell Death Assays

A variety of different parameters can be monitored to detect cell deathand apoptosis. Examples of such parameters include, but are not limitedto, monitoring activation of cellular pathways for toxicologicalresponses by gene or protein expression analysis, DNA fragmentation;changes in the composition of cellular membranes, membrane permeability,activation of components of death-receptors or downstream signalingpathways (e.g., caspases), generic stress responses, NF-kappaBactivation and responses to mitogens. Specific examples of such assaysfollow.

Morphological Changes: Apoptosis in many cell types is correlated withaltered morphological appearances. Examples of such alterations include,but are not limited to, plasma membrane blebbing, cell shape change,loss of substrate adhesion properties. Such changes are readilydetectable with a light microscope. Cells undergoing apoptosis can alsobe detected by fragmentation and disintegration of chromosomes. Thesechanges can be detected using light microscopy and/or DNA or chromatinspecific dyes.

Altered Membrane Permeability: Often the membranes of cells undergoingapoptosis become increasingly permeable. This change in membraneproperties can be readily detected using vital dyes (e.g., propidiumiodide and trypan blue). Similarly, dyes can be used to detect thepresence of necrotic cells. For example, certain methods utilize agreen-fluorescent LIVE/DEAD Cytotoxicity Kit #2, available fromMolecular Probes. The dye specifically reacts with cellular aminegroups. In necrotic cells, the entire free amine content is available toreact with the dye, thus resulting in intense fluorescent staining. Incontrast, only the cell-surface amines of viable cells are available toreact with the dye. Hence, the fluorescence intensity for viable cellsis reduced significantly relative to necrotic cells (see, e.g.,Haugland, 1996 Handbook of Fluorescent Probes and Research Chemicals,6th ed., Molecular Probes, OR).

Dysfunction of Mitochondrial Membrane Potential: Altered or defectivemitochondrial activity can result in mitochondrial collapse called the“permeability transition” or mitochondrial permeability transition.Proper mitochondrial functioning requires maintenance of the membranepotential established across the membrane. Dissipation of the membranepotential prevents ATP synthesis and thus halts or restricts theproduction of a vital biochemical energy source. Consequently, a varietyof assays designed to assess toxicity and cell death involve monitoringthe effect of a test agent on mitochondrial membrane potentials or onthe mitochondrial permeability transition. One approach is to utilizefluorescent indicators (see, e.g., Haugland, 1996 Handbook ofFluorescent Probes and Research Chemicals, 6th ed., Molecular Probes,OR, pp. 266-274 and 589-594). Various non-fluorescent probes can also beutilized (see, e.g., Kamo et al. (1979) J. Membrane Biol. 49:105).Mitochondrial membrane potentials can also be determined indirectly frommitochondrial membrane permeability (see, e.g., Quinn (1976) TheMolecular Biology of Cell Membranes, University Park Press, Baltimore,Md., pp. 200-217). Further guidance on methods for conducting suchassays is provided in PCT publication WO 00/19200 to Dykens et al.

Caspase Activation: Some assays for apoptosis are based upon theobservation that caspases are induced during apoptosis. Induction ofthese enzymes can be detected by monitoring the cleavage ofspecifically-recognized substrates for these enzymes. A number ofnaturally occurring and synthetic protein substrates are known (see,e.g., Ellerby et al. (1997) J. Neurosci. 17:6165; Kluck, et al. (1997)Science 275:1132; Nicholson et al. (1995) Nature 376:37; and Rosen andCasciola-Rosen (1997) J. Cell Biochem. 64:50). Methods for preparing anumber of different substrates that can be utilized in these assays aredescribed in U.S. Pat. No. 5,976,822.

Cytochrome c Release: In healthy cells, the inner mitochondrial membraneis impermeable to macromolecules. Thus, one indicator of cell apoptosisis the release or leakage of cytochrome c from the mitochondria.Detection of cytochrome c can be performed using spectroscopic methodsbecause of the inherent absorption properties of the protein.Alternatively, the protein can be detected using standard immunologicalmethods (e.g., ELISA assays) with an antibody that specifically binds tocytochrome c (see, e.g., Liu et al. (1996) Cell 86:147).

Assays for Cell Lysis: When cells die they typically release a mixtureof chemicals, including nucleotides, and a variety of other substances(e.g., proteins and carbohydrates) into their surroundings. Some of thesubstances released include ADP and ATP, as well as the enzyme adenylatecyclase which catalyzes the conversion of ADP to ATP in the presence ofexcess ADP. Thus, certain assays involve providing sufficient ADP in theassay medium to drive the equilibrium towards the generation of ATPwhich can subsequently be detected via a number of different means. Onesuch approach is to utilize a luciferin/luciferase system that is wellknown to those of ordinary skill in the art in which the enzymeluciferase utilizes ATP and the substrate luciferin to generate aphotometrically detectable signal. Further details regarding certaincell lysis assays are set forth in PCT publication WO 00/70082.

C. Test Agents

A variety of different types of agents can be screened for the abilityto modulate the activity CB2. The agents can be agonists or antagonists.The agents can be include, for example, antibodies, peptides or smallmolecules, hormones, naturally occurring molecules, or molecules fromexisting repertoires of chemical compounds synthesized by thepharmaceutical industry. Combinatorial libraries can be produced formany types of compounds that can be synthesized in a step-by-stepfashion. Such compounds include polypeptides, beta-turn mimetics,polysaccharides, phospholipids, hormones, prostaglandins, steroids,aromatic compounds, heterocyclic compounds, benzodiazepines, oligomericN-substituted glycines and oligocarbamates. Large combinatoriallibraries of the compounds can be constructed by the encoded syntheticlibraries (ESL) method described in PCT Publications WO 95/12608, WO93/06121, WO 94/08051, 95/35503 and WO 95/30642. Peptide libraries canalso be generated by phage display methods. See, e.g., Devlin, WO91/18980. Compounds to be screened can also be obtained from theNational Cancer Institute's Natural Product Repository, Bethesda, Md.,as well as a number of other commercial sources. The agents to bescreened can also be agonist antibodies and antagonist antibodies. Ageneral review of methods for preparing libraries is provided by Dolleand Nelson (J. Combinatorial Chemistry 1: 235-282 (1999)).

The following examples are provided to illustrate certain aspects of themethods and compositions that are provided but should not be construedto limit the scope of the claimed invention.

EXAMPLE

I. Methods

Materials. Culture media and reagents were from Gibco (Invitrogen,France). Fetal calf serum was from J Bio Laboratories (France). Pooledhuman AB positive serum was supplied by the National Transfusion Center.N-acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin(AC-DEVD-AFC) fluorogenic substrate and NS 398 were from Biomol andPDGF-BB from Preprotech Inc (Tebu, France). 2′,7′-dichlorofluoresceindiacetate (DCFH-DA) was from Molecular Probes (Interchim, France),Z-val-Ala-Asp (OCH3)-Fluoromethyl ketone (ZVAD-fink) from R&D Systems,4,6-diamidino-2-phenylindole (DAPI) from Biovalley (France), CellTiter96 AQ_(ueous) One Solution reagent from Promega (France) and ApoptoticDNA Ladder Kit from Roche (Germany). N-acetyl-cysteine, (Sigma) wasdissolved in PBS and buffered with NaOH to pH 7.4 prior to use. [³⁵S]GTPγS was from ICN (France). EUK8 (see, e.g., Pucheu, S. (1996)Cardiovasc. Drugs Ther. 10:331-339) was kindly provided by Eukarion Inc(Bedford, USA). The rabbit anti-CB2 receptor antisera (raised againstresidues 20-33 of the human CB2 receptor) and CB2 blocking peptide(residues 20-33 of the human CB2 receptor, having the amino acidsequence NPMKDYMILSGPQK) were from Cayman (Spibio, France). SR 144528({N-[S)-endo-1,3,3,-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(choloro-3-methylphenyl)-1-(4-methyl-benzyl)-pyrazole-3-carboxamide)and SR 141716A (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-di-chlorophenyl)-4-methylpyrazol-3-carboxamidehydrochloride) (8) were kindly provided by Sanofi (Montpellier, France)(see, also EP 656354-A1). ACEA (arachidonyl-2′-chloroethylamide[N-(2-Chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide; see reference [5])and JWH-015([(2-Methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone]; seereference [5]) were purchased from Tocris (Fisher Bioblock, France).Δ9-tetrahydrocannabinol(THC)([6a,7,8,10a-Tetrahydro-6,6,9-trimethyl-3-(pentyl-5,5,5-d3)-6H-dibenzo(b,d)pyran-1-ol])was from Sigma (France).

Human liver specimen. Snap frozen surgical liver resections from 13patients (8 men, 5 women, mean age 55 years, 39-72 range years) wereretrospectively studied. Normal liver samples were collected from 3women undergoing hepatic resection for colorectal metastasis (n=3).Cirrhotic samples were obtained from 8 livers of patients undergoingliver transplantation and from 2 patients undergoing hepatic resectionfor hepatocellular carcinoma. Cirrhosis was consecutive to chronic HCV(n=1) or HBV (n=2) infections, primary biliary cirrhosis (n=1),alcoholic liver disease (n=4), or Wilson disease (n=1) and remainedcryptogenic in 1 case. This study conformed to the ethical regulationsimposed by French legislation.

Immunohistochemical detection of CB2 receptors in normal and cirrhoticlivers. Frozen sections (5-7 μm) were air-dried and fixed in ice-coldacetone for 10 minutes at −20° C. Non specific binding was blocked bypreincubating sections 1 hr at room temperature with 20% AB-human serumin 50 mM Tris-buffered saline (TBS) pH 7.6. Excess antiserum wasremoved, and sections were further incubated over night at 4° C. with arabbit polyclonal antisera to human CB2 receptor, diluted 1/350 inantibody diluent (Dakopatts, France). After rinsing 3 times in TBS,sections were incubated for 45 min at room temperature with mousemonoclonal anti-rabbit immunoglobulin G antibodies, diluted 1/50(Dakopatts, France), rinsed 3 times in TBS, further incubated for 30 minat room temperature with rabbit anti-mouse immunoglobulin antibodies(Dakopatts, France), diluted 1/50, and then processed using the alkalinephosphatase-anti-alkaline phosphatase (APAAP) complex immunoenzymaticmethod. Briefly, after washing 3 times in TBS, slides were incubated for30 min at room temperature with the APAAP complex, diluted 1/25. Afterrinsing twice in TBS pH 7.6 and once in TBS pH 8.2, sections wererevealed using naphthol AS-TR phosphate-Fast Red TR (Sigma, France) inthe presence of levamisole, in order to block endogenous phosphataseactivity. Slides were counterstained with aqueous haematoxylin. Toconfirm the specificity of the primary antibody, controls includedpreadsorption of the primary antibody with the corresponding syntheticpeptide (100 μg/ml, for 1 hr at room temperature) or omission of theprimary antibody.

In order to determine whether hepatic myofibroblasts express CB2protein, double immunostaining of CB2 and smooth muscle α-actin wasperformed. Sections were first processed for CB2 immunostaining using astandard three-stage biotin-streptavidin immunoperoxidase method.Briefly endogenous peroxidase was quenched by incubation of the acetonefixed sections in TBS/0.3% H₂O₂ for 30 min then washed in TBS. Nonspecific binding was blocked by preincubating sections 30 min withTBS/20% AB-human serum. Sections were then incubated for 15 min inavidin followed by 15 min in biotin (Vector Laboratories, Avidin/Biotinblocking kit), and further incubated over night at 4° C. with the antiCB2 antisera. Subsequently, sections were washed in TBS and incubatedsuccessively with the secondary antibody biotinylated goat anti-rabbit(Dakopatts) (1/500) and streptavidin-horseradish peroxidase complex(1/50) (Pierce, Perbio, Interchim, France), 30 min each. Peroxidaseactivity was revealed using metal-enhanced diaminobenzidine (DAB)substrate (Pierce). All steps were carried out at room temperatureunless otherwise mentioned. Immunostaining for smooth muscle α-actin wasthen processed using the APAAP method described above, with a 1/5000dilution of a monoclonal antibody to smooth muscle α-actin (Sigma,France). Slides were counterstained with aqueous haematoxylin. Singleand double staining were visualized by bright-field photomicrographs onan Axioplan microscope (Zeiss, Oberkochen, Germany), equipped with adigital imaging system (Hamamatsu 3CCD color camera, HamamatsuPhotonics, France).

Isolation and culture of human hepatic myofibroblasts. Human hepaticmyofibroblasts were obtained by outgrowth of explants prepared fromsurgical specimens of normal liver, as previously described [10]. Thisprocedure was performed in accordance with ethical regulations imposedby the French legislation. Cells were cultured in Dulbecco's modifiedEagle's medium (DMEM) containing 10% serum (5% fetal calf serum, and 5%pooled human AB-positive serum, DMEM 5/5) and were used between thethird and seventh passage. Experiments were performed on cells that weremade quiescent by a 48 hrs incubation in serum-free Waymouth medium. Themyofibroblastic nature of the cells was evaluated as previouslydescribed [9]. The cultures were found to express two markers of rathepatic myofibroblasts, fibulin-2 and interleukin-6, and not theprotease P100, a marker for rat hepatic stellate cells [9].

Culture of CHO cells overexpressing CB1 or CB2 receptors. Cells werekindly provided by Sanofi (Montpellier, France) and were cultured inDMEM medium containing 5% SVF and 20 ng/ml gentamicin. Experiments wereperformed on cells that were made quiescent by a 48 hrs incubation inserum-free Waymouth medium containing 20 ng/ml gentamicin.

RNA Preparation and RT-PCR. Total RNA was extracted from confluentquiescent cells in 100 mm dishes, using RNeasy kit (Qiagen, France).cDNA was synthesized from 2 μg of total RNA by reverse transcription for1 h at 37° C. using 200 units of M-MLV reverse transcriptase(Invitrogen, France), in a 20 μl reaction mixture containing 0.05 μg/μloligo (dT)₁₂₋₁₈ primers (Invitrogen, France), 0.5 mM dNTPs (Promega,France) and 10 mM dithiothreitol in first strand buffer (Invitrogen,France). To check for eventual genomic DNA contamination, RT controlswere performed in the same conditions without reverse transcriptase.PCRs were performed with 2 μl of the reverse transcription reactionusing 1.25 units of AmpliTaq Gold DNA polymerase (Applied Biosystems,France) and the corresponding buffer supplemented with 2 mM MgCl2, 0.2mM dNTPs, and 25 pmol of each primer in a total volume of 50 μl. 40 PCRcycles were carried out in a GeneAmp 2700 thermalcycler (AppliedBiosystems, France), each cycle consisting of denaturation at 95° C. for45 s, annealing at 58° C. for 45 s, and extension at 72° C. for 30 s,with the first cycle containing an extended denaturation period (10 min)for the activation of the polymerase and the last cycle containing anextended elongation period (10 min). Oligonucleotide primers (MWGBiotech, France) for CB2 were as follows: CB2 sense primer5′-TTTCCCACTGATCCCCAATG-3′ and CB2 antisense primer,5′-AGTTGATGAGGCACAGCATG-3′, and the predicted PCR product of 337 bp. PCRamplified products were analyzed on a 1.5% agarose gel and blotted ontoHybond-N+membrane (Amersham Pharmacia Biostech, France). After aprehybridization in a buffer containing 6×SSC, 5 mM EDTA pH 8, 5×Denhardt, 0.1% SDS and 0.1 mg/ml ssDNA, for 2 hrs at 42° C., themembrane was hybridized overnight at 42° C. in the same buffercontaining 50 ng of the CB2 oligonucleotide probe5′-GACCCTAGGGCTAGTGTTGGCTG-3′, labeled with [γ-³²P] adenosinetriphosphate, using T4 kinase (Invitrogen). After hybridization, theblot was washed twice in 0.1% SDS, 1×SSC for 30 min at room temperatureand analyzed by phospho-imager (Molecular Dynamics, France).

Immunocytochemical detection of CB2 receptor in human hepaticmyofibroblasts. Human hepatic myofibroblasts were seeded (1000/cm²) in35 mm dishes, grown in serum-containing medium for 24 hrs. Afterwards,immunocytochemistry was performed on cells made quiescent by a 48 hrsincubation in serum-free Waymouth medium. Briefly, cells washed with TBSand fixed in 4% paraformaldehyde for 10 min, after which they werewashed in TBS and subsequently incubated in TBS/20% AB-human serum for30 min at room temperature. Cells were then incubated with the anti-CB2antisera (dilution 1/400 in TBS/20% AB-human serum) for 3 hrs at roomtemperature and overnight at 4° C. in a humid chamber. After incubationwith the primary antibody, cells were rinsed extensively in TBS andincubated with a Cy3-conjugated goat anti-rabbit IgG (Sigma) (dilution1/500, in TBS/20% AB-human serum) at room temperature, in the dark, for30 min. Cells were then rinsed extensively in TBS, mounted inVectashield mounting medium (Vector Laboratories, Burlingame, Calif.),sealed and observed by fluorescence microscopy. To confirm thespecificity of the primary antibody, controls included preadsorptionwith the corresponding synthetic peptide (100 μg/ml, for 1 hr at roomtemperature) or omission of the primary antibody.

[³⁵S] GTPγS binding assay. Membranes were obtained from confluenthepatic myofibroblasts or from CHO cells overexpressing CB2 receptors(kindly provided by Sanofi, Montpellier, France) made quiescent byincubation in Waymouth medium without serum for 48 hrs, as described in[9], and frozen at −80° C. until use. [³⁵S] GTP γS binding was performedin the conditions described in [9]. Specific binding was calculated asthe difference in bound radioactivity in the presence or absence of 10μM unlabeled GTPγS, and did not exceed 10% of the total binding.

Apoptosis assays. All the following techniques for measuring apoptosiswere performed on non-confluent cells allowed to attach overnight inDMEM 5/5 and serum-starved for 48 hrs, as previously reported [10; 11].Nuclear morphology was assayed using DAPI staining. Cells (10,000/cm²)in Lab-Tek chamber slides (Nalge Nunc International) were treated withthe indicated effectors for 8 hrs, fixed in 2% paraformaldehyde, stainedwith DAPI and viewed under fluorescence microscopy (ZEISS).Caspase-3-like activity was assayed on cell lysates obtained as follows.After treatment of cells (200,000 cells in 60 mm dishes) for variousperiods of time with the indicated effectors, floating cells werecollected, centrifuged and the pellet lysed in 50 μl lysis buffercontaining 50 mM HEPES pH 7.4, 100 mM NaCl, 1% NP-40, 1 mM EDTA (pH8.0), 1 mM DTT, Leupeptin 2 μg/ml, Aprotinin 2 μg/ml and pepstatin 1μg/ml. Adherent cells were lysed for 10 min on ice, in 0.2 ml lysisbuffer. The lysates from adherent and floating cells were pooled,centrifuged and the supernatant stored at −80° C. until use. DEVDaseactivity was measured in 200 μl assay buffer, containing 100 mM HEPES pH7.4, 10% sucrose, 10 mM DTT, 500 μM EDTA, 50 μg protein and 20 μMAC-DEVD-AFC as fluorogenic substrate. After 3 hrs at 37° C., thefluorescence of the reaction mixture was determined with aspectrofluorometer (FL600 Microplate Fluorescence Reader (BIO-TEK,France), with excitation and emission wavelengths of 400 nm and 530 nm,respectively. DNA laddering was assayed by agarose gel electrophoresisof total DNA extracted from cells (500,000 cells in 3 dishes of 100 mm)treated for 20 hrs with the indicated effectors. Total DNA wasextracted, using the Apoptotic DNA Ladder Kit according to themanufacturer's instructions, and was further incubated with 20 μg/mlRNase (DNase free) for 20 min at room temperature. Two μg of DNA waselectrophoresed on a 2% agarose gel stained with SYBR Green I, andanalyzed by phospho-imager (Molecular Dynamics, France).

Fluorescent measurement of intracellular reactive oxygen species. Thefluorescent probe DCFH-DA (dissolved at 5 mM in absolute ethanol) wasused for the assessment of intracellular reactive oxygen species (ROS).DCF fluorescence was measured using a FL-600 multiplate fluorimeter(Biotek Instruments, France), as previously described [11]. Cells (7,000cells in 96-well plates) were allowed to attach overnight in DMEM 5/5,and serum-starved for 2 days in DMEM without phenol red. Cells were thenloaded for 20 min at 37° C. with 5 μM of DCFH-DA in PBS and THC. Aftertwo washes in PBS, the fluorescence was monitored using excitation andemission wavelengths of 485 and 530 nm, respectively. Values werecorrected for hMF autofluorescence.

Cell viability. Cells (7,000 cells in 96-well plates) were allowed toattach overnight in DMEM5/5, serum-starved for 48 hrs in DMEM withoutphenol red and treated with the indicated effectors for 16 hrs.CellTiter 96 AQueous One Solution reagent was added to each well andabsorbance was recorded at 490 nm.

DNA synthesis. DNA synthesis was measured in triplicate wells byincorporation of [³H] thymidine, as previously described [12]. ConfluenthMF were made quiescent by incubation in Waymouth medium without serumfor 48 hrs. Cells were then stimulated for 30 hrs with 20 ng/ml PDGF-BB.[³H] thymidine (0.5 μCi/well) was added during the last 20 hrs ofincubation.

Statistics. Results are expressed as mean±S.E.M of n experiments.Results were analyzed by repeated measures two-way analysis of variance(ANOVA) followed by paired comparison corrected according to theBonferroni method. p<0.05 was taken as the minimum level ofsignificance.

II. Results

CB2 Receptors are Induced in Myofibroblastic Cells of Human CirrhoticLiver

CB2 receptor expression was studied by immunohistochemistry with apolyclonal antibody directed against human CB2 receptor, on frozentissue sections prepared from surgical samples of normal (n=3) andcirrhotic livers (n=10) (FIGS. 1A-1C). CB2 receptors were not detectedin normal liver (FIG. 1A). In contrast, cirrhotic samples showed astrong CB2 immunostaining of numerous spindle-shaped cells in fibroticsepta, irrespective of the etiology of cirrhosis (FIG. 1B). CB2 receptorexpression was also found in non-parenchymal cells, as well as ininflammatory cells and bile duct epithelial cells located along fibroticsepta. Specificity of the antibody was demonstrated by the lack ofsignal in slides incubated in the presence of the CB2 blocking peptide(FIG. 1B, inset) or by omitting the first antibody (not shown).

Double immunohistochemistry, using and anti-CB2 receptor antibody and analpha-smooth muscle actin antibody, clearly identified hepaticmyofibroblasts within fibrotic septa as a major cell type expressing CB2receptors (FIG. 1C). Accordingly, CB2 receptors were also expressed incultured human hepatic myofibroblasts, as demonstrated both by RT-PCRanalysis (FIG. 2A) and by immunocytochemistry (FIG. 2B).

The functionality of CB2 receptors expressed in human hepaticmyofibroblasts was studied in [³⁵S] GTPγS binding assays, which measureGDP-GTP exchange on the α (alpha) subunit of the G protein, and reflectthe initial steps of G protein activation by a receptor ligand. CHOcells overexpressing CB2 receptors served as controls. As shown in FIG.2C, Δ9-tetrahydrocannabinol (THC), a natural cannabinoid that binds toboth CB1 and CB2 receptors, dose-dependently increased binding of [³⁵S]GTPγS to G proteins in membranes of human hepatic myofibroblasts, withan EC50 of 10 nM and a maximal effect observed at 100 nM. In CHO-CB2overexpressing cells, the EC50 was 1 nM, and maximal activation occurredat 10 nM (FIG. 2C). The specific CB2 agonist JWH-015, which selectivelybinds to CB2 receptors with a potency similar to that of THC, was asefficient as THC in enhancing GTPγS binding (FIG. 2C, inset).

Antiproliferative Effect of CB2 Receptors in Human HepaticMyofibroblasts

THC dose-dependently inhibited DNA synthesis elicited by 20 ng/mlPDGF-BB (FIG. 3A), with a maximal 50% reduction of growth at 500 nM THC,half maximal inhibition occurring in the presence of 200 nM of thecompound.

We further defined the receptor involved, owing to the use of selectiveCB2 agonists (JWH-015 and cannabidiol) and of a selective CB1 agonist(ACEA). Specificity of the compounds for their respective receptors wasverified by assessing their effects in CHO cells overexpressing CB1 orCB2 receptors. In CHO overexpressing CB2 receptors, JW-015 stronglydecreased forskolin-stimulated cAMP levels, while ACEA had no effect;conversely, in CHO overexpressing CB1 receptors, ACEA reducedforskolin-stimulated cAMP levels, whereas JW-015 had no effect (FIG. 3A,inset). In human hepatic myofibroblasts, the selective CB2 receptoragonist JWH-015 was as potent as THC in inhibiting DNA synthesis (IC50150 nM), inducing a maximal 60% reduction of PDGF-BB-stimulated DNAsynthesis at 500 nM. In contrast, the CB1 agonist ACEA had no effect(FIG. 3A). In keeping with these results, pretreatment of cells with theCB2 receptor antagonist SR 144528 strongly reduced the antiproliferativeeffect of THC (FIG. 3B). These results demonstrate that the growthinhibitory effects of THC rely on CB2 receptor activation.

Activation of CB2 Receptors Induce Apoptosis of Human HepaticMyofibroblasts

THC also elicited cytotoxic effects towards serum-deprived human hepaticmyofibroblasts, as shown by cell rounding, shrinkage and detachment(FIG. 4A). Maximal decrease in cell survival was observed at 2 μM THC, aconcentration higher than that required to trigger growth inhibition.Several lines of evidence indicated that reduced viability was relatedto an apoptotic process. DAPI staining showed that cells exposed to THCexhibited condensed nuclei in contrast to control cells (FIG. 4B).Consistently, THC induced a time-dependent activation of caspase-3, witha maximal 6.7-fold±1.3 increase in activity peaking after 4-6 hr (FIG.4C); as expected, the general caspase inhibitor ZVAD-fink totallyblunted caspase-3 stimulation by THC (not shown). Finally, THC-treatedcells showed dramatic DNA laddering on agarose gel electrophoresis, incontrast to control serum-deprived cells which displayed intact DNA(FIG. 4D). ZVAD-fmk also blocked THC-induced DNA laddering (FIG. 4D).

Further experiments investigated whether THC-induced apoptosis wasreceptor-mediated. THC elicited a dose-dependent decrease in cellviability, as assessed by the MTS assay, half maximal inhibitionoccurring with a 1.5 μM concentration of the compound (FIG. 5A). JWH-015also dose-dependently reduced hepatic myofibroblast viability, halfmaximal effect occurring at 1 μM concentrations of the compound (FIG.5A). The cytotoxic effect of THC was blunted in cells pretreated withthe selective CB2 receptor antagonist SR 144528 (FIG. 6B), andaccordingly, cells exposed to SR 144528 showed no activation ofcaspase-3 in response to THC (FIG. 5, inset).

Molecular Mechanisms Mediating THC-Induced Growth Inhibition andApoptosis.

We investigated the molecular mechanism of THC-induced growth inhibitionof human hepatic myofibroblasts and focused on the inducible form ofcyclooxygenase, cyclooxygenase-2 (COX-2), since we previously showedthat COX-2 induction is a major antiproliferative pathway in humanhepatic myofibroblasts [13; 14; 9]. We investigated whether COX-2 mightalso be involved in cannabinoid elicited growth inhibition, owing to theuse of NS-398, a selective COX-2 inhibitor. As shown in FIG. 6A, NS-398reduced the antiproliferative effect of THC. Accordingly, THC caused astrong induction of COX-2, (FIG. 6B). In contrast ibuprofen or NS-398did not affect THC-induced caspase-3 (FIG. 6 c).

We recently unraveled oxidative stress as a mediator of apoptosiselicited by 15-d-prostaglandin J2 [11]. Therefore, subsequentexperiments were designed in order to investigate whether CB2-dependentapoptosis relies on oxidative stress. Two antioxidants were used, EUK 8,a superoxide dismutase and catalase mimetic with potent antioxidanteffects, and the glutathione precursor N-Acetyl Cysteine (NAC). Bothcompounds blunted activation of caspase-3 elicited by THC (FIG. 6C).Accordingly, treatment of cells with 3 μM THC increased by 1.5 fold theproduction of intracellular reactive oxygen species, as assessed withthe peroxide-sensitive probe DFCH-DA. (FIG. 6D). In contrast, EUK 8 orNAC did not affect THC-induced growth inhibition (FIG. 6A).

Taken together, these results demonstrate that growth inhibitory andapoptotic effects of CB2 receptors depends on distinct signallingpathways, induction of COX-2 and intracellular oxidative stress,respectively.

III. Discussion

Proliferation of myofibroblasts is central for the development of liverfibrosis during chronic liver diseases. We provide here the firstevidence that CB2 receptors are induced in hepatic myofibroblasts ofpatients with cirrhosis of various etiologies. Moreover, our resultsdemonstrate that activation of CB2 receptors reduce accumulation ofhuman hepatic myofibroblasts by eliciting growth inhibitory andapoptotic effects.

Data concerning expression and function of CB receptors in liver arescarce. It was previously shown that normal liver does not express CB2receptor mRNA. Accordingly, we did not detect CB2 byimmunohistochemistry in normal human liver samples. In contrast, we showthat CB2 receptors are strongly induced in the cirrhotic liver ofvarious etiologies and are expressed in non parenchymal cells andbiliary cells located within and at the edges of fibrotic septa. Doubleimmunohistochemistry identified hepatic myofibroblasts as a major celltype expressing CB2 receptors during chronic liver diseases.Heterogeneity of liver fibrogenic cells has recently been documented,with the description of at least two populations of smooth muscleα-actin myofibroblasts with fibrogenic potential, myofibroblastichepatic stellate cells and hepatic myofibroblasts, both of whichaccumulate during chronic liver injury (15, 16). Our experiments wereperformed with cultured hepatic myofibroblasts (smooth muscle alphaactin and fibulin-2 positive cells) and confirmed the expression of CB2mRNA and protein in these fibrogenic cells. Moreover, GTPγS bindingexperiments demonstrated that CB2 receptors are functional. Indeed, THCinduced activation of G protein with an affinity of 10 nM, in keepingwith the apparent affinity of THC for its receptors. In addition,JWH-015, the selective CB2 agonist, also elicited G protein activation,with a potency similar to that of THC. Although CB2 receptors arepredominantly expressed in spleen, tonsils and immune cells, they havealso been detected at low levels in various tissues. Interestingly, thepresence of CB2 receptors has also been described in mesangial cells, acell type that contributes to the pathogenesis of renal matrixaccumulation during chronic glomerular injury.

The ability of cannabinoids to control cell death and proliferation hasbeen well documented. Thus, antitumoral properties of cannabinoids havebeen linked to their growth inhibitory and apoptotic effects. Indeed,activation of CB2 receptors induce apoptosis of glioma cells in cultureand regression of malignant glioma in vivo. Cannabinoids also inducecell death in PC-12 pheochromocytoma cells, tumorigenic epidermal cells,and K-ras-transformed epithelial cells, via either CB1 or CB2-dependentpathways (for a review, see 17). Moreover, proliferation of breast andprostate cancer cells is strongly reduced by cannabinoids. We showherein that, in human hepatic myofibroblasts, CB2 triggers two majorantifibrogenic properties of human hepatic myofibroblasts, growthinhibition and apoptosis. These effects are elicited by differentcannabinoid concentrations, since submicromolar concentrations of THCinhibited growth without affecting cell viability, whereas apoptosisonly occurred at micromolar concentrations. Such dose-dependentselectivity of antiproliferative and apoptotic effects has previouslybeen described. Thus, in fetal hepatocytes, growth inhibition andapoptosis require different concentrations of TGF-β[18]. Similarly, lowconcentrations of two lipids, 15-d-PGJ2 and S1P are growth inhibitoryfor human hepatic myofibroblasts, whereas higher doses are apoptotic[19; 11; 9; 10]. Nevertheless, both THC-mediated growth arrest and celldeath of human hepatic myofibroblasts were CB2 receptor-dependent, asshown by the use of selective CB2 agonists and antagonists. Thus, botheffects were blocked by the CB2 receptor antagonist SR144528, andreproduced by the CB2 agonists cannabidiol and JW-015, but not by theCB1 agonist ACEA. In several studies, similar micromolar concentrationsof cannabinoids were also required to induce receptor-mediatedapoptosis, in particular in leukemia and lymphoma cells (19), or inhuman breast cancer cells (20). The reason as to why such concentrationsof THC are required to induce cell death are undefined, but CB2 receptordimerization could provide an explanation. Indeed, it is wellestablished that homo- or heterodimerization of G protein-coupledreceptors leads to increased or decreased potency of agonists to inducefunctional responses. Thus, orexin 1/CB1 receptor dimers show enhancedefficacy for orexin 1-mediated ERK activation. In contrast, neurotensinreceptor 1 and 3 complexes show decreased potency of neurotensin forneurotensin receptor 1-mediated functional responses. Furtherinvestigations are warranted to determine whether CB2 receptors can formhomo or heterodimers in human hepatic myofibroblasts.

Characterization of the molecular mechanisms underlying growth arrestand apoptosis following CB2 activation revealed distinct, nonoverlapping signalling pathways. Antiproliferative effects of THC relyon COX-2 induction and apoptosis is mediated by oxidative stress. Inhuman breast cancer cells, the growth inhibitory effect of anandamidewas consecutive to inhibition of cyclic AMP (cAMP) levels. However, thismechanism is unlikely in human hepatic myofibroblasts, since we haveshown that cAMP is an antiproliferative messenger [13; 14; 9], and cAMPlevels are not raised in response to THC in human hepatic myofibroblasts(not shown). We show that, in human hepatic myofibroblasts, growthinhibition by cannabinoids is tightly controlled by cyclooxygenases, therate-limiting enzymes in the conversion of arachidonic acid intoprostaglandins and thromboxanes. We previously showed that COX-2, theinducible form of cyclooxygenase, is central in growth inhibition ofhuman hepatic myofibroblasts. We described that endothelin-1, TNF-α andsphingosine-1-phosphate inhibit proliferation of these cells through apathway that involves induction of COX-2. We also showed that themitogenic effects of PDGF-BB and thrombin result from a balance betweena promitogenic and a COX-2-dependent growth inhibitory pathway. Althougha few studies suggested that cannabinoids stimulate arachidonic acidrelease in cultured astrocytes and neuroblastoma cells, and increasePGE2 production in the brain, induction of COX-2 by cannabinoids wasdescribed only in neuroglioma cells and lung cancer cells (39, 40).However, these studies did not investigate the biological consequencesof the production of arachadonic metabolites. We show here that NS-398,a selective COX-2 inhibitor, reduces the growth inhibitory effect ofTHC, demonstrating that the antiproliferative effects of cannabinoidsrely on COX-2 induction. Accordingly, THC triggers induction of COX-2.Therefore, the present data further support a central role of COX-2 inhuman hepatic myofibroblast growth inhibition, although additionalmechanisms may be involved. The consequences of COX-2 induction by THCmay involve regulation of more distal events, such as cell cyclecomponents.

We also demonstrate that apoptosis elicited by THC relies on anintracellular pathway distinct from that involved in growth inhibition.Indeed, blocking COX-2 by NS398 did not affect cell death triggered byTHC, suggesting the involvement of other signaling pathways. Incontrast, the antioxidants NAC and EUK 8, a superoxide/catalase mimeticdecrease the apoptotic response to THC (FIG. 7) without affecting growthinhibition elicited by the compound. These results indicate thatTHC-dependent apoptosis is mediated by oxidative stress, a resultfurther supported by the finding that apoptotic doses of THC alsostimulates ROS production (FIG. 7). Production of ROS occurs rapidly,being observed within 20 min exposure to THC, and therefore appears asan early signaling event in the apoptotic signaling pathway. Increasingevidences suggest a major role for ROS as intermediates for apoptosissignaling. Thus, production of ROS leads to growth inhibition andapoptosis of tumor and hematopoietic cells, in response to HGF, TNF-α,or Fas ligand. The signaling events initiated by ROS following THCstimulation and leading to human hepatic myofibroblast apoptosis areunder current investigation. Ceramide might be a possible candidate,since it triggers apoptosis in human hepatic myofibroblasts, and isassociated with apoptosis in response to cannabinoids in different celltypes.

Accumulation of hMF is one of the hallmarks of the fibrogenic process inthe liver and several lines of evidence indicate that limitingproliferation of these cells or inducing their apoptosis may representpromising therapeutic approaches. Along these lines, it was recentlydemonstrated in a model of chronic tetrachloride intoxication thatwithdrawal of the insulting agent is followed by apoptotic removal ofhMF and resolution of fibrosis. The causal link between hMF apoptosisand reversibility of fibrosis was further reinforced in a studydemonstrating that administration of gliotoxin induces apoptosis ofactivated MF in vitro and in vivo, reduces progression of fibrosisduring tetrachloride intoxication and enhances regression of fibrosisduring the recovery phase. The balance between pro and anti-apoptoticfactors for hMF may therefore be critical in the progression of liverfibrosis. This was recently exemplified with TIMP-1, a strong survivalfactor for activated MF, as shown by the fact that prolonged expressionof TIMP-1 is associated to a decreased spontaneous resolution of liverfibrosis. We show that CB2 receptors are not detected in the normalliver and are strongly induced in hepatic myofibroblasts in thecirrhotic liver. These results indicate that activation of CB2 receptorsduring chronic liver disease can limit progression of fibrosis.Interestingly, there appears to be a general up-regulation of thecannabinoid system during chronic liver diseases. Indeed, it wasrecently shown that there is an increased production of endogenousanandamide by monocytes of patients with chronic liver diseases which isassociated to up-regulation of CB1 receptors in vascular endothelialcells. These alterations elicit an increase in peripheral vasodilationand contribute to the pathogenesis of portal hypertension throughactivation of CB1 receptors.

To briefly summarize, immunohistochemistry experiments indicated that,in the normal liver, CB1 receptors were expressed around vessels,whereas CB2 receptors were not or faintly detected. However, humanhepatic myofibroblasts express the receptors for cannabinoids CB1 andCB2. In biopsies from patients with chronic liver diseases, CB1 and CB2receptors were detected in non parenchymal cells, in the lobule and inthe fibrous septa. CB1 and CB2 receptor mRNAs and proteins were alsodetected in cultured human hepatic myofibroblasts, as detected by RT-PCRand immunocytochemistry. Δ9-tetrahydrocannabinol, the main activecomponent of marijuana, reduced viability of serum-deprived hepaticmyofibroblasts by an apoptotic process, characterized by condensednuclei, fragmented DNA and increased caspase-3 activity.

Apoptosis involved CB receptors since the CB1/CB2 agonists CP 55940,HU-210, WIN-55.212-2, as well as anandamide were also cytotoxic.Apoptosis was triggered by CB2 receptors since i) the CB1 agonist ACEAwas not cytotoxic, whereas the CB2 agonist JWH-015 was apoptotic, andii) the CB2 antagonist SR 144528 prevented apoptosis elicited by HU-210,whereas the CB1 antagonist SR 141716 had no effect.

In conclusion, activation of CB2 leads to potent apoptosis of humanhepatic myofibroblasts and may therefore limit their accumulation duringliver fibrosis. The data presented herein show CB2 receptors as a novelantifibrogenic pathway in hMF. This pathway is up-regulated in cirrhoticpatients, indicating that a CB2-based antifibrotic strategy can be used,which is devoid of non desired CB1-mediated psychotropic andvasodilating effects.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent or patent application were specifically andindividually indicated to be so incorporated by reference.

References

-   1. Friedman, S. L. (2000) J Biol Chem 275, 2247-2250-   2. Piomelli, D., Giuffrida, A., Calignano, A., and Rodriguez de    Fonseca, F. (2000) Trends Pharmacol Sci 21, 218-224.-   3. Kumar, R. N., Chambers, W. A., and Pertwee, R. G. (2001)    Anaesthesia 56, 1059-1068.-   4. Bifulco, M., Laezza, C., Portella, G., Vitale, M., Orlando, P.,    De Petrocellis, L., and Di Marzo, V. (2001) Faseb J 29, 29-   5. Pertwee, R. G. (1999) Curr Med Chem 6, 635-664.-   6. Guzman, M., and Sanchez, C. (1999) Life Sci 65, 657-664-   7. Batkai, S., Jarai, Z., Wagner, J. A., Goparaju, S. K., Varga, K.,    Liu, J., Wang, L., Mirshahi, F., Khanolkar, A. D., Makriyannis, A.,    Urbaschek, R., Garcia, N., Jr., Sanyal, A. J., and Kunos, G. (2001)    Nat Med 7, 827-832-   8. Rinaldi-Carmona, M., Barth, F., Millan, J., Derocq, J. M.,    Casellas, P., Congy, C., Oustric, D., Sarran, M., Bouaboula, M.,    Calandra, B., Portier, M., Shire, D., Breliere, J. C., and Le    Fur, G. L. (1998) J Pharmacol Exp Ther 284, 644-650-   9. Davaille, J., Gallois, C., Habib, A., Li, L., Mallat, A., Tao,    J., Levade, T., and Lotersztajn, S. (2000) J Biol Chem 275,    34628-34633-   10. Davaille, J., Li, L., Mallat, A., and Lotersztajn, S. (2002) J    Biol Chem 277, 37323-37330.-   11. Li, L., Tao, J., Davaille, J., Feral, C., Mallat, A., Rieusset,    J., Vidal, H., and Lotersztajn, S. (2001) J Biol Chem 276,    38152-38158.-   12. Tao, J., Mallat, A., Gallois, C., Belmadani, S., Mery, P. F.,    Nhieu, J. T., Pavoine, C., and Lotersztajn, S. (1999) J Biol Chem    274, 23761-23769-   13. Mallat, A., Gallois, C., Tao, J., Habib, A., Maclouf, J.,    Mavier, P., Preaux, A. M., and Lotersztajn, S. (1998) J Biol Chem    273, 27300-27305-   14. Mallat, A., Fouassier, L., Preaux, A. M., Gal, C. S., Raufaste,    D., Rosenbaum, J., Dhumeaux, D., Jouneaux, C., Mavier, P., and    Lotersztajn, S. (1995) J Clin Invest 96, 42-49-   15. Knittel, T., Kobold, D., Saile, B., Grundmann, A., Neubauer, K.,    Piscaglia, F., and Ramadori, G. (1999) Gastroenterology 117,    1205-1221-   16. Cassiman, D., and Roskams, T. (2002) J Hepatol 37, 527-   17. Guzman, M., Sanchez, C., and Galve-Roperh, I. (2001) J Mol Med    78, 613-625-   18. Sanchez, A., Alvarez, A. M., Benito, M., and Fabregat, I. (1996)    J Biol Chem 271, 7416-7422-   19. McKallip, R. J., Lombard, C., Fisher, M., Martin, B. R., Ryu,    S., Grant, S., Nagarkatti, P. S., and Nagarkatti, M. (2002) Blood    100, 627-634-   20. De Petrocellis, L., Melck, D., Palmisano, A., Bisogno, T.,    Laezza, C., Bifulco, M., and Di Marzo, V. (1998) Proc Natl Acad Sci    USA 95, 8375-8380-   21. Ramer, R., Brune, K., Pahl, A., and Hinz, B. (2001) Biochem    Biophys Res Commun 286, 1144-1152-   22. Gardner, B., Zhu, L. X., Sharma, S., Tashkin, D. P., and    Dubinett, S. M. (2003) Faseb J 17, 2157-2159.-   23. Mechoulam et al. (1995) Biochem. Pharmacol. 50:83.

1. A method of treatment of a disease of the liver which involves theuse of cannabinoids, wherein the disease is selected from the groupconsisting of liver fibrosis, alcoholic liver cirrhosis, chronic viralhepatitis, non-alcoholic steatohepatitis and primary liver cancer.
 2. Amethod of treatment of a disease of the liver which involves the use ofagonists of CB2 receptor, wherein the disease is selected from the groupconsisting of liver fibrosis, alcoholic liver cirrhosis, chronic viralhepatitis, non-alcoholic steatohepatitis and primary liver cancer.
 3. Amethod of treatment of a disease of the liver which involves theactivation of CB2 receptors, wherein the disease is selected from thegroup consisting of liver fibrosis, alcoholic liver cirrhosis, chronicviral hepatitis, non-alcoholic steatohepatitis and primary liver cancer.4. A method of treatment of a disease of the liver which involves theup-regulation of CB2 receptors, wherein the disease is selected from thegroup consisting of liver fibrosis, alcoholic liver cirrhosis, chronicviral hepatitis, non-alcoholic steatohepatitis and primary liver cancer.5. A method of treating a disease of the liver, comprising administeringan effective amount of a cannabinoid to a patient having the disease,wherein the disease is selected from the group consisting of liverfibrosis, alcoholic liver cirrhosis, chronic viral hepatitis,non-alcoholic steatohepatitis and primary liver cancer.
 6. A method oftreating a disease of the liver, comprising administering an agent thatactivates a CB2 receptor to a patient having the disease, wherein thedisease is selected from the group consisting of liver fibrosis,alcoholic liver cirrhosis, chronic viral hepatitis, non-alcoholicsteatohepatitis and primary liver cancer.
 7. A method of treating adisease of the liver, comprising administering a composition comprisinga non-selective agonist of CB2 and a selective antagonist of CB1 to apatient having the disease, wherein the disease is selected from thegroup consisting of liver fibrosis, alcoholic liver cirrhosis, chronicviral hepatitis, non-alcoholic steatohepatitis and primary liver cancer.